EP3649452B1 - Préparation d'échantillon en phase gazeuse pour microscopie cryo-électronique - Google Patents
Préparation d'échantillon en phase gazeuse pour microscopie cryo-électronique Download PDFInfo
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- EP3649452B1 EP3649452B1 EP18828718.9A EP18828718A EP3649452B1 EP 3649452 B1 EP3649452 B1 EP 3649452B1 EP 18828718 A EP18828718 A EP 18828718A EP 3649452 B1 EP3649452 B1 EP 3649452B1
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- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Definitions
- X-ray crystallography has traditionally been the standard for providing structural analysis for biological research, and while X-ray crystallography still provides high resolution structural information, this technique has several downsides.
- One such limitation is that a large amount of sample is required for X-ray crystallography. This makes X-ray crystallography impractical in cases where it is difficult to generate large, highly pure quantities of the target molecule.
- cryo-EM Single particle cryo-electron microscopy
- Document US 2013/205 808 A1 discloses a method for preparing a sample for cryo-electron microscopy wherein a combined vapor and sample beam is directed at a substrate surface.
- sample vitrification is far from ideal.
- Typical sample preparation techniques involve solubilization of protein analytes in water followed by pipetting onto a hydrophilic EM grid. The grid is blotted with filter paper (removing >99.99% of the sample) and then plunged into a bath of cryogen, vitrifying the remaining water/sample.
- a key component to obtaining proper 3D data from EM structures is that the particles must all be of the same structural conformation but randomly oriented within the amorphous ice. Proper 3D analysis is completed via reconstruction of a number of images, thus requiring numerous particles randomly oriented in the same structural conformation.
- This process can also impart a preferred orientation, especially if the molecules are thicker in one dimension than another.
- data acquisition times must be extended and data files become very large (i.e., greater than 5Tb), since much of the EM grid must be imaged to generate sufficient signal for analysis.
- conventional approaches puts a significant strain on the computational resources required to analyze the data Cheng et al., Cell, 2015, 161 (3):438-449 ).
- cryo-EM sample preparation methods and systems so as to improve the applicability and accuracy of cryo-EM.
- the present invention provides a method for preparing a sample for cryo-electron microscopy (cryo-EM) according to claim 1 and a cryo-electron microscopy (cryo-EM) sample preparation system according to claim 12.
- formation of an amorphous solid layer is independent of the deposition of a sample to be analyzed, thus allowing for the generation of uniformly thick layer.
- the formation of an amorphous solid layer may be monitored during formation and any imperfection in the solid layer may be corrected using ion milling or related techniques.
- the present invention is also able to provide cryo-EM sample preparation resulting in increased image resolution, decreased image acquisition time, and increased sensitivity.
- Certain aspects of the invention further include the use of mass spectrometry to purify analyte particles, including but not limited to proteins, protein complexes, and cells, in the gas-phase for subsequent vitrification.
- Samples prepared in this way can be extracted from the mass spectrometer using a cryo-transfer sample holder and placed directly into an EM for imaging.
- One implementation of this method utilizes a modified mass spectrometer that allows for gas-phase purification of analyte ions. The ions will be passed over a cooled sample probe where they are deposited onto an EM sample holder and vitrified.
- Amorphous solids, or non-crystalline solids refer to solids that lack the long-range molecular order characteristic of crystals.
- ice formed using the methods and systems described herein is preferably vitreous ice (also referred to herein as amorphous ice).
- Common H 2 O ice is a hexagonal crystalline material where the molecules are regularly arranged in a hexagonal lattice.
- vitreous ice lacks the regularly ordered molecular arrangement.
- Vitreous ice and the other amorphous solids available with the present invention are produced either by rapid cooling of the liquid phase (so the molecules do not have enough time to form a crystal lattice) or by compressing ordinary ice (or ordinary solid forms) at very low temperatures.
- One embodiment of the present invention provides a method for preparing a sample for cryo-electron microscopy (cryo-EM) comprising the steps of forming a vapor stream of atoms or molecules, and directing the vapor stream toward a substrate surface such that the atoms or molecules impinge on the substrate surface while under vacuum.
- the substrate surface is at a temperature of -100°C or less (optionally at a temperature of -150°C or less, -175°C or less, or -195°C or less).
- a layer of an amorphous solid is formed on the surface of the substrate.
- the method further comprises forming an analyte beam containing charged or uncharged analyte particles to be analyzed using EM; and contacting the amorphous solid layer with the analyte beam.
- the analyte particles are embedded on or within the deposited amorphous solid layer thereby forming a suitable sample for EM analysis.
- under a vacuum refers to a pressure of 10 -4 Torr or less, a pressure 10 -5 Torr or less, or a pressure 10 -6 Torr or less.
- the atoms or molecules of the vapor stream contact the substrate surface, and the step of contacting the amorphous solid layer with the analyte beam, are carried out at a pressure equal to or less than 10 -4 Torr, 10 -5 Torr, or 10 -6 Torr.
- the analyte particles forming the analyte beam can be charged or uncharged particles depending on the deposition method used to deposit the molecules onto the ice layer.
- the analyte particles and the molecules making the amorphous solid layer are substantially randomly orientated when deposited on the substrate, such as on a membrane, film, or EM grid.
- the analyte beam can be an ion beam, molecular beam, or particle beam.
- the analyte particles are ions formed using techniques including, but not limited to, electrospray ionization and laser desorption, such as matrix-assisted laser desorption/ionization (MALDI).
- MALDI matrix-assisted laser desorption/ionization
- the analyte particles are ionized under native electrospray conditions so as not to perturb structural conformation of the particles.
- the analyte ions are formed using a mass spectrometer which optionally isolates or purifies the analyte ions.
- the particle beam is a molecular beam.
- the molecular beam is produced by creating an aerosol of an analyte particle containing solution and introducing the aerosol into the vacuum system.
- the analyte beam is characterized by an intensity selected from the range of 0.025 to 25 particles per 1 ⁇ m 2 per second, 0.05 to 10 particles per 1 ⁇ m 2 per second, or 0.1 to 5 particles per 1 ⁇ m 2 per second. In certain embodiments, the analyte beam is characterized by a spot size selected from the range of 800 ⁇ m 2 to 3.8E7 ⁇ m 2 .
- the analyte particles can be purified or isolated, such as by a mass spectrometer device, before being deposited onto the amorphous solid.
- the analyte beam is characterized by a purity of at least 85%, 90%, 95%, or 99%.
- analyte particles such as proteins, which may have significant conformational structures
- it is desirable that the analyte beam is characterized by a conformation purity of at least 85%, 90%, 95% or 99%.
- Analyte particles useful with the present invention include, but are not limited to, protein molecules, multi-protein complexes, protein/nucleic acid complexes, nucleic acid molecules, virus particles, micro-organisms, sub-cellular components (e.g., mitochondria, nucleus, Golgi, etc.), and whole cells.
- the analyte particles are molecular entities, single molecules, or multiple molecules complexed together through non-covalent interactions (such as hydrogen bonds or ionic bonds).
- the analyte particles have a molecular mass exceeding 1,000 Daltons, 10,000 Daltons, 50,000 Daltons, 100,000 Daltons, or 150,000 Daltons.
- a further embodiment comprises additionally contacting the amorphous solid layer with the atoms or molecules from the vapor stream after the analyte particles have been deposited on the amorphous solid layer. This will provide an additional layer of the solid on top of the deposited molecules.
- a further method comprises contacting the amorphous solid layer with the analyte beam concurrently with contacting the substrate surface with the atoms or molecules from the vapor stream.
- the vapor stream (or molecular water beam) is reflected off of one or more reflecting surfaces prior to contacting the substrate surfaced. This ensures that the atoms or molecules are broken up and have a randomized orientation before contacting the substrate.
- the vapor stream is generated using a Knudsen-type effusion cell, a molecular beam doser, or a co-effusion of a matrix with analyte into the system.
- the vapor stream is characterized by an intensity selected from the range of 4.8E9 to 2.8E11 molecules per ⁇ m 2 per second and/or a spot size selected from the range of 800 ⁇ m 2 to 3.8E7 ⁇ m 2 .
- the vapor stream comprises a flux of molecules having a uniformity within 98% over an area of 7 mm 2 .
- the vapor stream is a molecular beam.
- Molecular beams are streams of molecules traveling in the same or similar direction and can be produced by allowing a gas at higher pressure to expand through a small orifice into a chamber at lower pressure to form the beam.
- the incident trajectory of the particles of the vapor stream contacting the substrate surface is within 1 degree of normal to the substrate surface.
- the vapor stream is controlled, or the deposited amorphous solid layer is milled, etched, or otherwise refined, so that the amorphous solid layer has a thickness of 2 microns or less, 150 nm or less, or 100 nm or less.
- the amorphous solid layer has a uniform thickness which does not vary by more than 5% across the substrate.
- the layer of the amorphous solid has an extent of crystallinity less than or equal to 1%.
- the layer of the amorphous solid has a purity of at least 85%, 90%, 95%, or 99%.
- the vapor stream can comprise any molecules or atoms able to form amorphous solids where exposed to extremely low temperatures and pressures.
- molecules and atoms include, but are not limited to, cyclohexanol, methanol, ethanol, isopentane, water, O 2 , Si, SiO 2 , S, C, Ge, Fe, Co, Bi and mixtures thereof.
- the vapor stream comprises charged molecules.
- the vapor stream comprises water molecules.
- an embodiment of the present invention provides a method for preparing a sample for cryo-electron microscopy (cryo-EM) comprising the steps of forming a molecular beam of water molecules, and contacting a surface of a substrate with the molecular water beam while under vacuum.
- the substrate surface is at a temperature of -100°C or less (optionally at a temperature of -150°C or less, -175°C or less, or -195°C or less).
- a layer of amorphous ice is formed on the surface of the substrate.
- the method further comprises forming an analyte beam containing charged or uncharged analyte particles to be analyzed using EM; and contacting the deposited ice layer with the analyte beam.
- the molecular beam is controlled, or the deposited ice is milled, etched, or otherwise refined, so that the deposited ice layer with the analyte particles has a thickness of 2 microns or less, preferably 150 nm or less, or 100 nm or less.
- the ice layer has a uniform thickness which does not vary by more than 5% across the substrate.
- the analyte particles are ions and the analyte source is able to generate a controllable ion beam containing charged analyte ions (such as electrospray ion deposition) and direct the ion beam to contact the receiving surface of the cryo-EM probe.
- the system further comprises a modified mass spectrometer that can provide purified ions to the analyte source.
- the system comprises an electron microscope where the cryo-EM probe is directly transferred from the deposition portion of the instrument to the microscope portion of the instrument for analysis.
- the formation of the vitreous ice layer or other amorphous solid layer is monitored (alone or in conjunction with the deposition of the analyte particles) to ensure that the proper thickness is achieved and that the layer deposited on the substrate is amorphous as opposed to crystalline.
- a microscale is utilized to confirm that a solid (such as ice) is being uniformly deposited on the substrate, continuous film, continuous membrane, cryo-EM grid or probe.
- the vacuum chamber contains a window or other means to allow optical light or infrared light to illuminate the sample.
- An optical or infrared light detection cell able to receive light transmitted or reflected from the deposited solid layer is then used to determine if the solid is amorphous or crystalline.
- the monitoring of the formation of the amorphous solid is performed in real time.
- the present invention provides a cryo-electron microscopy (cryo-EM) sample preparation system comprising: a) a vacuum chamber; b) a cryo-EM probe positioned with the vacuum chamber, wherein the cryo-EM probe comprises a receiving surface; c) a beam doser able to produce a controllable molecular beam (or vapor stream) and direct the molecular beam (or vapor stream) to contact the receiving surface of the cryo-EM probe; d) a temperature control means able to provide a temperature of -100°C or less to the receiving surface of the cryo-EM probe; and e) an analyte source in fluid communication with the vacuum chamber, wherein the analyte source is able to produce a controllable analyte beam containing charged or uncharged analyte particles and direct the analyte beam to contact the receiving surface of the cryo-EM probe.
- cryo-EM cryo-electron microscopy
- the molecular beam comprises cyclohexanol, methanol, ethanol, isopentane, water, O 2 , Si, SiO 2 , S, C, Ge, Fe, Co, Bi, or mixtures thereof.
- the beam doser is able to produce a controllable molecular water beam.
- the vacuum chamber is able to provide a pressure of 10 -4 Torr, a pressure of 10 -5 Torr, or a pressure of 10 -6 Torr.
- the temperature control means is able to provide a temperature of -150°C or less, -175°C or less, or -195°C or less.
- the temperature control means comprises a cold finger able to provide localized temperature control of the receiving surface of the cryo-EM probe.
- the substrate described in the embodiments provided herein is an electron microscopy (EM) grid as known in the art.
- the EM grid may comprise a metal, including but not limited to copper, rhodium, nickel, molybdenum, titanium, stainless steel, aluminum, gold, or combinations thereof as known in the art.
- the EM grid may comprise a continuous film or membrane which is positioned across the top or bottom surface of the grid, or within the holes of the grid, so as to provide a solid support for the formation of the amorphous solid.
- the EM grid is covered by a thin film or membrane which includes, but is not limited to, films and membranes comprising graphene, graphene oxide, silicon oxide, silicon nitride, carbon, and combinations thereof.
- the molecular beam intended to form the amorphous solid may pass through at least a portion of the holes in the grid without producing a suitable layer.
- the film or membrane should be thin enough so as to not scatter electrons.
- the film or membrane has an approximate thickness or 15 nm or less, 10 nm or less, 5 nm or less, 2 nm or less, or 1 nm or less.
- the substrate is an EM grid comprising a graphene or graphene oxide monolayer film or membrane positioned across the surface of the grid.
- An embodiment of the present invention provides a method for forming a layer of vitreous ice on a substrate comprising the steps of: a) forming a molecular beam of water molecules; b) while under vacuum (i.e., at a pressure of 10 -4 Torr or less, a pressure 10 -5 Torr, or less or a pressure 10 -6 Torr or less), contacting the substrate with the molecular water beam, wherein the substrate is at a temperature of -100°C or less (optionally at a temperature of -150°C or less, -175°C or less, or -195°C or less), thereby forming a layer of vitreous ice on the substrate.
- the deposited ice layer has a thickness of 2 microns or less, 150 nm or less, or 100 nm or less.
- the present invention provides novel cryo-EM sample preparation methods that utilize in vacuo vitrification able to: (1) increase image resolution, (2) decrease image acquisition time, and (3) allow for many orders of magnitude increase in sensitivity.
- readily available mass spectrometry instruments can be used to purify proteins and protein complexes in the gas-phase for subsequent in vacuo vitrification. Samples prepared in this way can be extracted from the mass spectrometer - using a cryo-transfer sample holder and placed directly into an EM for imaging.
- Figs. 1-5 and 14-16 show exemplary cryo-electron microscopy (cryo-EM) sample preparation systems 25 according to certain embodiments of the present invention.
- a cryo-EM probe 2 able to hold or contain a sample is inserted into vacuum chamber 1.
- the temperature of the system is maintained using a coolant, such as liquid nitrogen, which is stored in tank 8 and transferred through cold finger 5, while one or more turbo pumps 9 are used to maintain the vacuum.
- a coolant such as liquid nitrogen
- Analyte particles are collected in an analyte source 6 where they are focused into an analyte beam 13 (such as through electrospray ion deposition) and directed to contact the sample plate being held by cryo-EM probe 2 (see Fig. 2 ).
- Fig. 3 shows one type of an analyte source 6 where analyte ions or molecules are drawn into the analyte source 6 through capillary 16.
- One or more ion optic devices such as skimmers 17, are used to focus the analyte ions into a beam 13 and to control the release speed of the ions through exit aperture 18.
- a beam doser 4 is used to generate one or more 14 molecular beams (such as molecular water beams) and direct the molecular beam down to the cryo-EM probe 2.
- Vapor 34 (such as water vapor) used to generate the molecular beams 14 is transported through a heated transfer line 19 and then transported along the same axis as travelled by the analyte beam 13.
- the moleculer beams 14 are reflected off a series of reflecting surfaces 26, which breaks up the molecules and randomizes their orientation (see Fig. 4 , panel b).
- Microchannel plate 20 only allows reflected molecular beams 14 to pass through if the beams are orientated along the proper direction (i.e., in a direction co-axial with the analyte beam 13). It should be noted that while these examples specify water vapor and
- the cryo-EM probe 2 is used to laterally move the sample as needed.
- the sample holder 21 can move the probe 2 to a quartz crystal microscale 10 which is used to monitor the build-up of the ice layer on the cryo-EM probe 2 (see Figs. 1 and 5 ).
- the sample can be moved to an infrared (IR) sample plate 15 which is illuminated by an IR light beam 22 provided by a fiber optic IR light source 7.
- the transmitted light is collected by the optical detection cell 11 and transmitted to the fiber optic IR spectrometer 23 to monitor whether the deposited ice layer comprises vitreous ice or crystalline ice.
- Figs. 14-16 illustrate a cryo-electron microscopy (cryo-EM) sample preparation system 25 where the system comprises an a secondary pumping system 27 used for making the vapor stream 34, and a storage tank 28 able to directly store the sample from the vacuum chamber 1 after the vitreous ice has been formed on the sample.
- the storage tank is kept low temperatures and can also utilize coolants such as liquid nitrogen.
- Protein ions can be purified in the gas-phase, collected in vacuo , and, once removed from the vacuum, retain their enzymatic function ( Blake et al., Analytical Chemistry, 2004, 76(21):6293-6305 ). Following these experiments, a mass spectrometer was modified so that analyte protein ions could be purified and deposited directly onto a sample probe.
- the probe surface originally comprised glycerol on stainless steel; however, with the requested cryo-transfer probe, the purified protein ions are deposited directly onto a cryogenic EM grid that has previously been covered in vitreous ice. Formation of the ice layer independent of the sample allows for the generation uniformly thick ice. Any imperfection in the ice may be corrected using ion milling or related techniques. Independently forming the ice also allows for appropriate quality control measures prior to committing the sample.
- Vitreous ice undergoes a phase transition from high density amorphous (HDA) ice at atmospheric pressure where it is formed, to low density amorphous (LDA) ice at the low pressures of the mass spectrometer ( Mishima et al., Nature, 1998, 396(6709): 329-335 ). It is essential that the purified protein ions have structures that are reflective of that from solution. Ion mobility experiments have shown that under native spray conditions this is achieved ( Seo et al., Angewandte Chemie-International Edition, 2016, 55(45): 14173-14176 ). Additionally, ion/ion chemical reactions are also used to reduce the charge of a purified protein population prior to deposition, and thereby restore solution phase structure.
- HDA high density amorphous
- LDA low density amorphous
- the present invention provides methods and instruments for preparing samples of an analyte with amorphous solids for use in cryo-electron microscopy (cryo-EM).
- the amorphous solids which protect the analyte from radiation damage and dehydration during imaging, must remain transparent to the electron beam during EM. This requires the amorphous solid layer (e.g., the ice layer) be thin, on the order of the same thickness of the molecules to be analyzed, and the solid must be amorphous. If the amorphous solid becomes too thick, the electrons may be scattered causing defocusing and reduction in image contrast.
- vitreous ice plays in the outer solar system and interstellar space ( Fama et al., Surface Science, 2008, 602(1): 156-161 ; and Cleeves et al., Science, 2014, 345(6204): 1590-1593 ).
- the high vacuum and coldness of space provides a natural forming ground for vitreous ice.
- it is the most common form of ice outside our solar system ( Guillot et al., J. Chemical Physics, 2004, 120(9):4366-4382 ).
- the obvious difficulties of studying vitreous ice have required the development of techniques to form amorphous ice in the laboratory under interstellar conditions, in particular, in cryogenic vacuum chambers.
- amorphous solids in conjunction with mass spectrometry instruments and techniques as described herein provide a novel means of preparing vitreous samples for cryo-EM which removes all the limitations associated with the currently employed plunge freezing method.
- an uncovered cryo-EM grid or a cryo-EM grid covered by a thin continuous film or membrane held at liquid nitrogen temperatures is used as a landing surface within a mass spectrometer.
- Examples of EM grids with continuous films or membranes across the surface of the grid can be obtained from Quantifoil Micro Tools GmbH (Großlöbichau, Germany) and Electron Microscopy Sciences (Hatfield, Pennsylvania, USA).
- the grid (covered or uncovered) is populated with biomolecules utilizing gas-phase analyte purification techniques described above.
- a molecular beam doser aimed at the landing surface.
- the job of the doser is to produce a controllable molecular beam of water which impacts the cryo-surface/grid, forming vitreous ice (for general descriptions of dosers, see Guillot et al., J. Chemical Physics, 2004, 120(9):4366-4382 ; Moeller et al., Optical Engineering, 2012, 51(11): 115601 ; and Westly et al., J. Chemical Physics, 1998, 108(8):3321-3326 ).
- analyte particles either directly from an analyte source or after gas-phase purification using a mass spectrometer. Concomitantly with the collection of the analyte particles, the molecular water beam is used to encase the analyte particles in amorphous ice. Alternately, the analyte particles are landed on an initial amorphous ice surface and then covered/encased with amorphous ice using the vapor stream.
- Figs. 6-9 show transmission electron microscopy (TEM) images of amorphous ice formed on a graphene oxide support film supported by copper and/or gold grids.
- the vitreous ice was collected at a temperature of -175°C, with a 15 minute exposure to the molecular beam doser under vacuum.
- the resulting ice layer was approximately 15 microns thick.
- Hexagonal ice was obtained the same manner, with the exception that the temperature was -155°C.
- holes 40 in the support grid covered by regions of amorphous ice 41 can be clearly distinguished from regions or holes in the grid without any formation of ice 42. Holes 47 were also drilled through the amorphous ice regions 41 crystalline ice regions 43 with an electron beam.
- amorphous ice regions 41 appear very different from the regions of crystalline hexagonal ice 43.
- these figures demonstrate that a layer of a solid was formed over the holes in the grid, where the layer was not the typical crystalline form of the solid.
- Fig. 11 shows IR spectra of amorphous H 2 O ice and crystalline H 2 O ice deposited on a substrate ( Mastrapa et al., Icarus, 2008, 197:307-320 ). As can be seen in these spectra, the peaks from the amorphous ice shifted to shorter wavelengths compared to the peaks from the crystalline ice.
- Fig. 12 shows IR spectra of crystalline hexagonal ice (bottom line) and amorphous ice (top line) using the present invention.
- Fig. 13 illustrates the rate of growth of the amorphous ice by measuring the frequency of a quartz crystal microbalance.
- the physical characteristics of the quartz crystal microbalance resulted in a 21 Hz decrease in resonance frequency for every 1 nm thickness of ice which forms on its surface. Accordingly, from 20 minutes to 50 minutes, the rate of ice formation in this experiment was 1.94 nm/min
- Fig. 10 shows a grid 44 without the use of a continuous film or membrane. Water molecules could simply pass through at least a portion of the holes in the grid. As a result, there may be little or no ice bridging the empty holes 46. However, a layer of ice spanning several holes 45 was still seen.
- the vitreous ice which protects analytes from radiation damage and dehydration during imaging, must remain transparent to the electron beam during EM. This requires the ice layer be thin, on the order of the same thickness of the molecules to be analyzed, and the ice must be amorphous. If the amorphous ice becomes too thick, the electrons may be scattered causing defocusing and reduction in image contrast. If crystalline ice begins to form, the electrons will be diffracted and the resulting diffraction pattern will obscure the image ( Cheng et al., Cell, 2015, 161(3): 438-449 ).
- amorphous solids In addition to water, there are other substances which will form amorphous solids at cold temperatures. These include, but are not limited to cyclohexanol, methanol, ethanol, isopentane, O 2 , SiO 2 , S, C, Ge, Fe, Co, and Bi, among many others. As with water, the amorphous state is obtained through condensation from the gas phase. Unlike water, which can be transformed to an amorphous solid by several techniques, these elements require vapor-condensation to form in the non-crystalline state ( Zallen R., The Physics of Amorphous Solids, 1983, 8-10 ).
- a vapor stream of the matrix in question is formed by heating (thermal vaporization), vaporization by electron beam, vaporization by ion bombardment, or by plasma-induced decomposition, all in vacuum.
- the vacuum chamber contains a cold surface onto which the atoms condense. Their thermal energy is extracted before they can migrate to the crystalline conformation. The result is a thin film ( ⁇ 50 micron thick) of amorphous solid.
- Example 1 The fact that substances such as (but not limited to) Si, Ge, Fe, Co, and Bi require vapor condensation to form, the incorporation of these compounds with the instrument of Example 1 provides a novel means of preparing vitreous samples for cryo-EM with matrices other than ice.
- the porosity/density of the amorphous material being formed can be controlled through the deposition angle employed as well as the energy of the deposited matrix molecules ( Dohnalek et al., Journal of Chemical Physics, 2003, 118(1): 364-372 ). This novel capability will enable fine tuning of the amorphous material to provide maximum protection to the biomolecules during cryo-EM analysis.
- a cryo-EM grid (either uncovered or a grid covered by a thin film or membrane) held at liquid nitrogen temperatures is used as a landing surface within the sample preparation instrument (Example 1).
- the grid is populated with biomolecules utilizing electrospray deposition.
- a vaporization source aimed at the landing surface. This source may be placed off axes to effect the angle of incidence of the matrix molecules on the surface.
- the job of the vaporization source is to produce a controllable vapor stream of material (including but not limited to H 2 O, Si, Ge, Fe, Co, Bi) that impacts the cryo-surface/grid, forming and amorphous solid.
- a very thin layer of material is generated on the grid. This is followed by collection of macromolecules isolated by the ESI deposition source. Concomitantly with the collection of these molecules, the vapor stream is used to encase the sample. When collection/sample preparation is complete, the probe and EM grid are removed from the sample preparation instrument and transferred directly into the cryo-EM.
- compositions are synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
- consisting of excludes any element, step, or ingredient not specified in the claim element.
- consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or elements.
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Claims (16)
- Procédé de préparation d'un échantillon pour cryo-microscopie électronique (cryo-EM), comprenant les étapes consistant à :a) former un courant de vapeur (34) d'atomes ou de molécules ;b) diriger le courant de vapeur (34) vers une surface de substrat de telle sorte que les atomes ou les molécules frappent la surface de substrat tout en étant sous vide, la surface de substrat étant à une température de -100°C ou moins, formant ainsi une couche solide amorphe d'atomes ou de molécules sur la surface de substrat, la couche solide amorphe ayant une épaisseur de 2 microns ou moins ;c) former un faisceau d'analyte (13) contenant des particules d'analyte chargées ou non chargées ; etd) mettre en contact la couche solide amorphe avec le faisceau d'analyte (13), incorporant ainsi les particules d'analyte sur ou dans la couche solide amorphe déposée.
- Procédé selon la revendication 1, comprenant en outre la mise en contact de la couche solide amorphe avec des atomes ou des molécules provenant du courant de vapeur (34) après que les particules d'analyte ont été incorporées sur la couche solide amorphe, ou
la mise en contact de la surface de substrat avec le faisceau d'analyte (13) en même temps que la mise en contact de la surface de substrat avec les atomes ou les molécules provenant du courant de vapeur (34). - Procédé selon l'une des revendications 1 et 2, dans lequel le courant de vapeur (34) comprend des molécules ou des atomes capables de former des solides amorphes, lesdites molécules ou lesdits atomes comprenant un ou plusieurs parmi le cyclohexanol, le méthanol, l'éthanol, l'isopentane, l'eau, O2, Si, SiO2, S, C, Ge, Fe, Co et Bi.
- Procédé selon l'une des revendications 1 à 3, dans lequel le substrat est une grille pour microscopie électronique (ME) comprenant un film ou membrane continu(e) positionné(e) sur une surface supérieure ou inférieure de la grille pour ME.
- Procédé selon l'une des revendications 1 à 4, dans lequel les atomes ou les molécules du courant de vapeur (34) entrent en contact avec la surface de substrat à une pression égale ou inférieure à 10-4 Torr, et/ou le faisceau d'analyte entre en contact avec la couche solide amorphe à une pression égale ou inférieure à 10-4 Torr.
- Procédé selon l'une des revendications 1 à 5, comprenant en outre l'étape d'analyse desdites particules d'analyte sur ou dans la couche solide amorphe à l'aide d'un cryo-microscope électronique.
- Procédé selon l'une des revendications 1 à 6, dans lequel le faisceau d'analyte (13) est un faisceau d'ions généré à l'aide d'une ionisation par électronébulisation ou d'une désorption laser, un faisceau moléculaire produit par création d'un aérosol d'une solution contenant des particules d'analyte et introduction dudit aérosol dans le système à vide, ou un faisceau de particules.
- Procédé selon l'une des revendications 1 à 6, dans lequel le courant de vapeur (34) est généré à l'aide d'une cellule d'effusion de type Knudsen, un doseur de faisceau moléculaire (4) ou une co-effusion d'une matrice avec analyte dans le système.
- Procédé selon l'une des revendications 1 à 8, comprenant en outre la surveillance de la formation de la couche solide amorphe en temps réel et/ou le fraisage ou la gravure de la surface de la couche solide amorphe pour retirer des difformités dans la couche.
- Procédé selon l'une des revendications 1 à 9 comprenant en outre la réflexion des atomes ou des particules du courant de vapeur sur une ou plusieurs surfaces réfléchissantes avant la mise en contact avec la surface du substrat.
- Procédé selon les revendications 1 à 3 dans lequel le courant de vapeur (34) est un faisceau moléculaire comprenant des molécules d'eau, et la couche solide amorphe est une couche de glace amorphe.
- Système (25) de préparation d'échantillon pour cryo-microscopie électronique (cryo-EM), comprenant :a) une chambre à vide ;b) une sonde de cryo-EM (2) positionnée dans la chambre à vide (1), ladite sonde de cryo-EM (2) comprenant une surface de réception ;c) un moyen de contrôle de la température, capable de fournir une température de -100°C ou moins à la surface de réception de la sonde de cryo-EM (2) ; caractérisé pard) un doseur de faisceau (4) capable de produire un faisceau moléculaire contrôlable et de diriger ledit faisceau moléculaire (14) pour entrer en contact avec la surface de réception de la sonde de cryo-EM (2) ;e) une source d'analyte en communication fluidique avec la chambre à vide, la source d'analyte (6) étant capable de produire un faisceau d'analyte contrôlable (13) contenant des particules d'analyte chargées ou non chargées et de diriger ledit faisceau d'analyte pour entrer en contact avec la surface de réception de la sonde de cryo-EM (2).
- Système selon la revendication 12, dans lequel le moyen de contrôle de température comprend un doigt froid (5) capable de fournir un contrôle de température localisé de la surface de réception de la sonde de cryo-EM (2).
- Système selon l'une des revendications 12 ou 13- dans lequel la source d'analyte (6) est capable de générer un faisceau d'ions contrôlable (13) contenant des ions d'analyte chargés et de diriger ledit faisceau d'ions pour entrer en contact avec la surface de réception de la sonde de cryo-EM (2) et/ou- comprenant un spectromètre de masse modifié capable de fournir des ions à la source d'analyte (6).
- Système selon l'une des revendications 12 à 14, dans lequel le doseur de faisceau (4) est capable de produire un faisceau d'eau moléculaire contrôlable.
- Système selon l'une des revendications 12 à 15, comprenant en outre :- une microbalance en contact avec la sonde de cryo-EM (2), la microbalance étant capable de mesurer la glace déposée sur la surface de réception de la sonde de cryo-EM (2), ou- une source lumineuse (7) capable d'éclairer une couche de glace déposée sur la surface de réception de la sonde de cryo-EM (2) et une cellule de détection optique ou de lumière ou infrarouge (11) capable de recevoir de la lumière transmise ou réfléchie par ladite couche de glace.
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EP3869182A1 (fr) * | 2020-02-19 | 2021-08-25 | FEI Company | Dispositif et procédé pour déterminer une propriété d'un échantillon destiné à être utilisé dans un microscope à particules chargées |
US11728146B2 (en) | 2021-01-13 | 2023-08-15 | Wisconsin Alumni Research Foundation | Retractable ion guide, grid holder, and technology for removal of cryogenic sample from vacuum |
US20240060863A1 (en) * | 2021-01-13 | 2024-02-22 | Wisconsin Alumni Research Foundation | Freezing and jacketing gas-phase biomolecules with amorphous ice for electron microscopy |
CN113484108B (zh) * | 2021-05-31 | 2022-03-11 | 中国科学院生物物理研究所 | 应用界面电荷制备冷冻电镜样品的方法 |
GB2614323B (en) * | 2021-12-30 | 2024-03-13 | Fei Co | A method and apparatus of preparing a sample of one or more molecule(s) for imaging with a cryo-electron microscope |
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US8884248B2 (en) * | 2012-02-13 | 2014-11-11 | Fei Company | Forming a vitrified sample for electron microscopy |
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